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US20110185448A1 - Markers associated with soybean rust resistance and methods of use therefor - Google Patents

Markers associated with soybean rust resistance and methods of use therefor Download PDF

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US20110185448A1
US20110185448A1 US13/054,760 US200913054760A US2011185448A1 US 20110185448 A1 US20110185448 A1 US 20110185448A1 US 200913054760 A US200913054760 A US 200913054760A US 2011185448 A1 US2011185448 A1 US 2011185448A1
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seq
sbr
soybean
marker
nos
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Ju-Kyung Yu
Becky Breitinger
Glenn Bowers
Nanda Chakraborty
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/54—Leguminosae or Fabaceae, e.g. soybean, alfalfa or peanut
    • A01H6/542—Glycine max [soybean]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

  • the presently disclosed subject matter relates to markers associated with soybean rust (SBR) resistance and methods of use therefor. More particularly, the presently disclosed subject matter relates to screening soybean lines for resistance to SBR and for producing soybean lines with improved resistance to SBR, the methods involving genetic marker analysis.
  • SBR soybean rust
  • Plant pathogens are known to cause massive damage to important crops, resulting in significant agricultural losses with widespread consequences for both the food supply and other industries that rely on plant materials. As such, there is a long felt need to reduce the incidence and/or impact of agricultural pests on crop production.
  • Soybean rust SBR
  • Phakopsora pachyrhizi H. Sydow & Sydow was first reported in Japan in 1902. By 1934, the pathogen was reported in several other Asian countries and in Australia. More recently, P. pachyrhizi infection has been reported in Africa, and has spread rapidly through the African continent.
  • SBR has the potential to cause significant yield losses in the U.S., as indicated by fungicide trials in Georgia and Florida that reported yield losses of 30 to 33% in untreated control plots.
  • the total yield loss in the 2006-2007 growing season due to SBR was estimated to be over U.S. $2.26 billion with an average of 2.3 fungicide applications required per season.
  • Yield losses up to 80% have been reported due to severe outbreaks of SBR, which result in early leaf drop that inhibits pod set. Consistent economic losses in Brazil over the last several years due to severe SBR outbreaks have raised concerns regarding the potential impact of this disease in the United States. Soybean cultivars currently available commercially in the United States are all susceptible to SBR to some degree, and fungicide applications are currently employed to control the disease.
  • soybean rust resistant cultivars are needed to reduce fungicide costs and yield losses due to SBR.
  • the presently disclosed subject matter provides methods for conveying resistance to soybean rust (SBR) into non-resistant soybean germplasm.
  • the methods comprise introgressing SBR resistance into a non-resistant soybean using one or more nucleic acid markers for marker-assisted breeding among soybean lines to be used in a soybean breeding program, wherein the markers are linked to an SBR resistance locus selected from the group consisting of Rpp1, Rpp2, Rpp3, Rpp4, and Rpp5.
  • the presently disclosed subject matter also provides methods for reliably and predictably introgressing soybean rust (SBR) resistance into non-resistant soybean germplasm.
  • the methods comprise employing one or more nucleic acid markers for marker-assisted breeding among soybean lines to be used in a soybean breeding program.
  • the presently disclosed subject matter also provides methods for producing a soybean plant adapted for conferring resistance to soybean rust (SBR).
  • the methods comprise (a) selecting a first donor parental line possessing a desired SBR resistance and having at least one of the resistant loci selected from a locus mapping to Rpp1 and mapped by one or more of the markers SEQ ID NOs: 1-3; a locus mapping to Rpp2 and mapped by one or more of the markers SEQ ID NOs: 4-6; a locus mapping to Rpp3 and mapped by one or more of the markers SEQ ID NOs: 7 and 8; a locus mapping to Rpp4 and mapped by one or more of the markers SEQ ID NOs: 9 and 10; and a locus mapping to Rpp5 and mapped one or more markers SEQ ID NOs: 11-13; (b) crossing the first donor parent line with a second parental line in hybrid combination to produce a segregating plant population; (c) screening the segregating plant population for identified chromosomal loci of
  • the presently disclosed subject matter also provides methods for selecting a soybean rust (SBR) resistant soybean plant.
  • the methods comprise (a) genotyping one or more soybean plants with respect to one or more single nucleotide polymorphisms (SNPs); and (b) selecting a soybean plant that includes at least one resistance allele associated with the SNPs, thereby selecting an SBR resistant soybean plant.
  • SNPs single nucleotide polymorphisms
  • the presently disclosed methods comprise (a) isolating one or more nucleic acids from a plurality of soybean plants; (b) detecting in said isolated nucleic acids the presence of one or more marker molecules associated with SBR resistance, wherein said marker molecule is selected from the group consisting of SEQ ID NOs: 1-13; and (c) selecting a soybean plant comprising said one or more marker molecules, thereby selecting an SBR resistant soybean plant.
  • the one or more nucleic acid markers are selected from the group consisting of SEQ ID NOs: 1-13, and informative fragments thereof.
  • the methods and compositions of the presently disclosed subject matter employ a marker molecule mapped within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 centiMorgans or less from a marker molecule selected from the group consisting of SEQ ID NOs: 1-13.
  • the marker-assisted breeding comprises single nucleotide polymorphism (SNP) analysis.
  • the methods further comprise screening an introgressed soybean plant, or a cell or tissue thereof, for SBR resistance.
  • the at least one resistance allele is associated with an allele having an A at nucleotide 428 of SEQ ID NO: 1; a T at position 895 of SEQ ID NO: 2; a G at position 932 of SEQ ID NO: 2; a T at position 57 of SEQ ID NO: 3; a G at position 213 of SEQ ID NO: 4; a G at position 441 of SEQ ID NO: 4; an A at position 70 of SEQ ID NO: 5; a T at position 348 of SEQ ID NO: 5; an A at position 715 of SEQ ID NO: 6; a C at position 377 of SEQ ID NO: 7; a T at position 100 of SEQ ID NO: 8; a G at position 113 of SEQ ID NO: 8; a T at position 147 of SEQ ID NO: 9; a C at position 205 of SEQ ID NO: 10; an A at position 102 at SEQ ID NO: 11; A T at position 159 of
  • the presently disclosed subject matter also provides soybean rust (SBR) resistant soybean plants, parts thereof (including but not limited to pollen, ovule, leaf, embryo, root, root tip, anther, flower, fruit, stem, shoot, seed; rootstock, protoplast, and callus), and progeny thereof, selected using the disclosed methods.
  • SBR soybean rust
  • FIGS. 1A-1E are genetic linkage maps depicting linkage groups and showing relative positions of various markers linked to Rpp1-Rpp5, respectively.
  • SEQ ID Nos: 1-13 are nucleotide sequences of the soybean genome comprising single nucleotide polymorphisms (SNPs) identified as being associated with the Rpp1 gene, the Rpp2 gene, the Rpp3 gene, the Rpp4 gene, and/or the Rpp5 gene as set forth in Table 1.
  • SNPs single nucleotide polymorphisms
  • SEQ ID NOs: 14-81 are nucleotide sequences of oligonucleotide primers that can be employed to amplify and/or otherwise assay a subsequence of the soybean genome that is associated with the Rpp1-Rpp5 loci as also set forth in Table 1.
  • SEQ ID NOs: 82-94 corresponds to subsequences of the preliminary assembly of the soybean ( Glycine max ) genomic sequence present in the Phytozyme Database, which correspond to SEQ ID NOs: 1-13 as set forth in Table 2.
  • the presently disclosed subject matter relates at least in part to the identification of several SNPs associated with SBR resistance in Glycine sp.
  • methods of conveying SBR resistance into non-resistant soybean germplasm which employ one or more of the identified SNPs in various approaches.
  • the articles “a”, “an”, and “the” refer to “one or more” when used in this application, including in the claims.
  • the phrase “a marker” refers to one or more markers.
  • the phrase “at least one”, when employed herein to refer to an entity refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more of that entity, including but not limited to whole number values between 1 and 100 and greater than 100.
  • the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments ⁇ 0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
  • allele refers to any of one or more alternative forms of a gene, all of which relate to at least one trait or characteristic. In a diploid cell, two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes, although one of ordinary skill in the art understands that the alleles in any particular individual do not necessarily represent all of the alleles that are present in the species. Since the presently disclosed subject matter relates to SNPs, it is in some instances more accurate to refer to a “haplotype” (i.e., an allele of a chromosomal segment) instead of “allele”. However, in such instances, the term “allele” should be understood to comprise the term “haplotype”.
  • the phrase “associated with” refers to a recognizable and/or assayable relationship between two entities.
  • a trait, locus, QTL, SNP, gene, marker, phenotype, etc. is “associated with resistance” if the presence or absence of the trait, locus, QTL, SNP, gene, marker, phenotype, etc., influences an extent or degree of resistance (e.g., resistance to SBR).
  • an allele associated with resistance to SBR comprises an allele having an A at nucleotide 428 of SEQ ID NO: 1; a T at position 895 of SEQ ID NO: 2; a G at position 932 of SEQ ID NO: 2; a T at position 57 of SEQ ID NO: 3; a G at position 213 of SEQ ID NO: 4; a G at position 441 of SEQ ID NO: 4; an A at position 70 of SEQ ID NO: 5; a T at position 348 of SEQ ID NO: 5; an A at position 715 of SEQ ID NO: 6; a C at position 377 of SEQ ID NO: 7; a T at position 100 of SEQ ID NO: 8; a G at position 113 of SEQ ID NO: 8; a T at position 147 of SEQ ID NO: 9; a Cat position 205 of SEQ ID NO: 10; an A at position 102 at SEQ ID NO: 11; A T at position 159 of SEQ ID NO: 12;
  • backcross refers to a process in which a breeder crosses a progeny individual back to one of its parents, for example, a first generation hybrid F1 with one of the parental genotypes of the F1 hybrid. In some embodiments, a backcross is performed repeatedly, with a progeny individual of one backcross being itself backcrossed to the same parental genotype.
  • chromosome is used herein in its art-recognized meaning of the self-replicating genetic structure in the cellular nucleus containing the cellular DNA and bearing in its nucleotide sequence the linear array of genes.
  • cultivar and “variety” refer to a group of similar plants that by structural or genetic features and/or performance can be distinguished from other varieties within the same species.
  • the term “gene” refers to a hereditary unit including a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a particular characteristics or trait in an organism.
  • hybrid in the context of plant breeding refers to a plant that is the offspring of genetically dissimilar parents produced by crossing plants of different lines or breeds or species, including but not limited to the cross between two inbred lines.
  • inbred refers to a substantially homozygous individual or line.
  • the phrase “informative fragment” refers to a nucleic acid molecule and/or its nucleotide sequence that allows for the proper identification of which allele of an allele set (e.g., an SNP) the nucleic acid molecule and/or the nucleotide sequence corresponds to
  • an SNP that corresponds to SEQ ID NO: 1 relates to an “A” or a “G” at position 428
  • an “informative fragment” of SEQ ID NO: 1 would be any sequence that comprises position 428 of SEQ ID NO: 1, thereby allowing the nucleotide that is present in that position to be determined.
  • introduction refers to both a natural and artificial process whereby genomic regions of one species, variety, or cultivar are moved into the genome of another species, variety, or cultivar, by crossing those species.
  • the process can optionally be completed by backcrossing to the recurrent parent.
  • linkage refers to a phenomenon wherein alleles on the same chromosome tend to be transmitted together more often than expected by chance if their transmission was independent.
  • two alleles on the same chromosome are said to be “linked” when they segregate from each other in the next generation less than 50% of the time, less than 25% of the time, less than 20% of the time, less than 15% of the time, less than 10% of the time, less than 5% of the time, less than 4% of the time, less than 3% of the time, less than 2% of the time, or less than 1% of the time.
  • two loci are linked if they are within 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 centiMorgans (cM) of each other.
  • cM centiMorgans
  • an SNP is linked to a marker if it is within 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cM of the marker.
  • linkage group refers to all of the genes or genetic traits that are located on the same chromosome. Within the linkage group, those loci that are close enough together can exhibit linkage in genetic crosses. Since the probability of crossover increases with the physical distance between loci on a chromosome, loci for which the locations are far removed from each other within a linkage group might not exhibit any detectable linkage in direct genetic tests.
  • linkage group is mostly used to refer to genetic loci that exhibit linked behavior in genetic systems where chromosomal assignments have not yet been made.
  • linkage group is synonymous with the physical entity of a chromosome, although one of ordinary skill in the art will understand that a linkage group can also be defined as corresponding to a region of (i.e., less than the entirety) of a given chromosome.
  • locus refers to a position that a given gene or a regulatory sequence occupies on a chromosome of a given species.
  • a marker refers to an identifiable position on a chromosome the inheritance of which can be monitored.
  • a marker comprises a known or detectable nucleic acid sequence.
  • a marker corresponds to an amplification product generated by amplifying a Glycine sp. nucleic acid with two oligonucleotide primers, for example, by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the phrase “corresponds to an amplification product” in the context of a marker refers to a marker that has a nucleotide sequence that is the same (allowing for mutations introduced by the amplification reaction itself) as an amplification product that is generated by amplifying Glycine sp. genomic DNA with a particular set of primers.
  • the amplifying is by PCR
  • the primers are PCR primers that are designed to hybridize to opposite strands of the Glycine sp. genomic DNA in order to amplify a Glycine sp. genomic DNA sequence present between the sequences to which the PCR primers hybridize in the Glycine sp. genomic DNA.
  • a marker that “corresponds to” an amplified fragment is a marker that has the same sequence of one of the strands of the amplified fragment.
  • the term “soybean” refers to a plant, or a part thereof, of the genus Glycine including, but not limited to Glycine max.
  • the phrase “soybean-specific DNA sequence” refers to a polynucleotide sequence having a nucleotide sequence homology of in some embodiments more than 50%, in some embodiments more than 60%, in some embodiments more than 70%, in some embodiments more than 80%, in some embodiments more than 85%, in some embodiments more than 90%, in some embodiments more than 92%, in some embodiments more than 95%, in some embodiments more than 96%, in some embodiments more than 97%, in some embodiments more than 98%, and in some embodiments more than 99% with a sequence of the genome of the species Glycine that shows the greatest similarity to it.
  • a “soybean-specific DNA sequence” can comprise a part of the DNA sequence of a soybean genome that flanks and/or is a part of an Rpp gene sequence.
  • molecular marker refers to an indicator that is used in methods for visualizing differences in characteristics of nucleic acid sequences.
  • indicators are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion and deletion mutations (INDEL), microsatellite markers (SSRs), sequence-characterized amplified regions (SCARs), cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of the markers described herein which defines a specific genetic and chromosomal location.
  • RFLP restriction fragment length polymorphism
  • AFLP amplified fragment length polymorphism
  • SNPs single nucleotide polymorphisms
  • INDEL microsatellite markers
  • SCARs sequence-characterized amplified regions
  • CAS cleaved amplified polymorphic sequence
  • nucleotide sequence homology refers to the presence of homology between two polynucleotides. Polynucleotides have “homologous” sequences if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence.
  • the “percentage of sequence homology” for polynucleotides can be determined by comparing two optimally aligned sequences over a comparison window (e.g., about 20-200 contiguous nucleotides), wherein the portion of the polynucleotide sequence in the comparison window can include additions or deletions (i.e., gaps) as compared to a reference sequence for optimal alignment of the two sequences.
  • a comparison window e.g., about 20-200 contiguous nucleotides
  • Optimal alignment of sequences for comparison can be conducted by computerized implementations of known algorithms, or by visual inspection. Readily available sequence comparison and multiple sequence alignment algorithms are, respectively, the Basic Local Alignment Search Tool (BLAST; Altschul et al.
  • an offspring plant refers to any plant resulting as progeny from a vegetative or sexual reproduction from one or more parent plants or descendants thereof.
  • an offspring plant can be obtained by cloning or selfing of a parent plant or by crossing two parent plants and include selfings as well as the F1 or F2 or still further generations.
  • An F1 is a first-generation offspring produced from parents at least one of which is used for the first time as donor of a trait, while offspring of second generation (F2) or subsequent generations (F3, F4, and the like) are specimens produced from selfings or crossings of F1s, F2s and the like.
  • An F1 can thus be (and in some embodiments is) a hybrid resulting from a cross between two true breeding parents (the phrase “true-breeding” refers to an individual that is homozygous for one or more traits), while an F2 can be (and in some embodiments is) an offspring resulting from self-pollination of the F1 hybrids.
  • phenotype refers to a detectable characteristic of a cell or organism, which characteristics are at least partially a manifestation of gene expression.
  • plant part refers to a part of a plant, including single cells and cell tissues such as plant cells that are intact in plants, cell clumps, and tissue cultures from which plants can be regenerated.
  • plant parts include, but are not limited to, single cells and tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, and seeds; as well as scions, rootstocks, protoplasts, calli, and the like.
  • population refers to a genetically heterogeneous collection of plants sharing a common genetic derivation.
  • the term “primer” refers to an oligonucleotide which is capable of annealing to a nucleic acid target, and serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of a primer extension product is induced (e.g., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH).
  • a primer in some embodiments an extension primer and in some embodiments an amplification primer
  • the primer is in some embodiments single stranded for maximum efficiency in extension and/or amplification.
  • the primer is an oligodeoxyribonucleotide.
  • a primer is typically sufficiently long to prime the synthesis of extension and/or amplification products in the presence of the agent for polymerization.
  • the minimum lengths of the primers can depend on many factors, including, but not limited to temperature and composition (A/T vs. G/C content) of the primer.
  • amplification primers these are typically provided as a pair of bi-directional primers consisting of one forward and one reverse primer or provided as a pair of forward primers as commonly used in the art of DNA amplification such as in PCR amplification.
  • primer can refer to more than one primer, particularly in the case where there is some ambiguity in the information regarding the terminal sequence(s) of the target region to be amplified.
  • a “primer” can include a collection of primer oligonucleotides containing sequences representing the possible variations in the sequence or includes nucleotides which allow a typical base pairing.
  • Primers can be prepared by any suitable method. Methods for preparing oligonucleotides of specific sequence are known in the art, and include, for example, cloning and restriction of appropriate sequences and direct chemical synthesis. Chemical synthesis methods can include, for example, the phospho di- or tri-ester method, the diethylphosphoramidate method and the solid support method disclosed in U.S. Pat. No. 4,458,066.
  • Primers can be labeled, if desired, by incorporating detectable moieties by for instance spectroscopic, fluorescence, photochemical, biochemical, immunochemical, or chemical moieties.
  • target polynucleotides can be detected by hybridization with a probe polynucleotide which forms a stable hybrid with that of the target sequence under stringent to moderately stringent hybridization and wash conditions. If it is expected that the probes are essentially completely complementary (i.e., about 99% or greater) to the target sequence, stringent conditions can be used. If some mismatching is expected, for example if variant strains are expected with the result that the probe will not be completely complementary, the stringency of hybridization can be reduced. In some embodiments, conditions are chosen to rule out non-specific/adventitious binding.
  • probe refers to a single-stranded oligonucleotide sequence that will form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence analyte or its cDNA derivative.
  • Rpp1 refers to loci that have been associated with SBR resistance as defined by the markers defined herein.
  • these loci are said to be present on Glycine linkage groups G, J, C2, G, and N, and linked to the markers depicted in FIGS. 1A-1E , respectively.
  • QTL quantitative trait locus
  • QTL plural quantitative trait loci
  • QTLs refers to a genetic locus (or loci) that controls to some degree a numerically representable trait that, in some embodiments, is continuously distributed.
  • QTL is used herein in its art-recognized meaning to refer to a chromosomal region containing alleles (e.g., in the form of genes or regulatory sequences) associated with the expression of a quantitative phenotypic trait.
  • a QTL “associated with” resistance to SBR refers to one or more regions located on one or more chromosomes and/or in one or more linkage groups that includes at least one gene the expression of which influences a level of resistance and/or at least one regulatory region that controls the expression of one or more genes involved in resistance to SBR.
  • QTLs can be defined by indicating their genetic location in the genome of a specific Glycine sp. accession using one or more molecular genomic markers. One or more markers, in turn, indicate a specific locus. Distances between loci are usually measured by the frequency of crossovers between loci on the same chromosome. The farther apart two loci are, the more likely that a crossover will occur between them.
  • centiM centiMorgan
  • the term “regenerate”, and grammatical variants thereof, refers to the production of a plant from tissue culture.
  • resistant encompass both partial and full resistance to infection (e.g., infection by a pathogen that causes SBR).
  • a susceptible plant can either be non-resistant or have lower levels of resistance to infection relative to a resistant plant.
  • the term is used to include such separately identifiable forms of resistance as “full resistance”, “immunity”, “intermediate resistance”, “partial resistance”, and “hypersensitivity”.
  • stringent hybridization conditions refers to conditions under which a polynucleotide hybridizes to its target subsequence, typically in a complex mixture of nucleic acids, but to essentially no other sequences. Stringent conditions are sequence-dependent and can be different under different circumstances. Exemplary guidelines for the hybridization of nucleic acids can be found in Tijssen (1993) in Laboratory Techniques in Biochemistry and Molecular Biology , Elsevier, New York, N.Y., United States of America; Ausubel et al. (1999) Short Protocols in Molecular Biology Wiley, New York, N.Y., United States of America; and Sambrook & Russell, 2001 (supra).
  • stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH.
  • Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).
  • Stringent conditions are those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C.
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary stringent hybridization conditions include: 50% formamide, 5 ⁇ SSC, and 1% SDS, incubating at 42° C.; or 5 ⁇ SSC, 1% SDS, incubating at 65° C.; with one or more washes in 0.2 ⁇ SSC and 0.1% SDS at 65° C.
  • a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures can vary between about 32° C. and 48° C. (or higher) depending on primer length.
  • the term “susceptible” refers to a plant having no resistance to the disease resulting in the plant being affected by the disease, resulting in disease symptoms.
  • the term “susceptible” is therefore equivalent to “non-resistant”.
  • the term “susceptible” can be employed in a relative context, in which one plant is considered “susceptible” because it is less resistant to a particular pathogen than is a second plant (which in the context of these terms in a relative usage, would be referred to as the “resistant” plant”).
  • Rpp?(Hyuuga) from the Japanese cultivar “Hyuuga” has been mapped by to the same region on LG C2 as Rpp3.
  • a fifth gene, Rpp5 was recently identified from PI200456 and mapped on LG N.
  • DOE-JGI U.S. Department of Energy-Joint Genome Institute
  • Rpp1 has been fine mapped to a 23 kb region on scaffold 21 of LG G, and several markers close to this gene have been identified (Hyten et al. (2008) Theor Appl Genet 116:945-952).
  • Rpp1, Rpp2, Rpp3, Rpp4, and Rpp5 can be race-specific, and can be overcome by various P. pachyrhizi isolates. For example, resistance in soybean lines carrying either Rpp1 or Rpp3 genes failed in Brazil within two years of the establishment of the disease.
  • the presently disclosed subject matter provides in some embodiments soybean varieties that are resistant to SBR, methods for identifying soybean plants that carry desirable resistance genes, and methods for introducing such desirable resistance genes into soybeans.
  • the presently disclosed subject matter provides for better models for marker-assisted breeding (MAB).
  • the presently disclosed subject matter therefore relates to methods of plant breeding and to methods to select plants, in particular soybean plants, particularly cultivated soybean plants as breeder plants for use in breeding programs or cultivated soybean plants for having desired genotypic or potential phenotypic properties, in particular related to producing valuable soybeans, also referred to herein as commercially valuable plants.
  • a cultivated plant is defined as a plant being purposely selected or having been derived from a plant having been purposely selected in agricultural or horticultural practice for having desired genotypic or potential phenotypic properties, for example a plant obtained by inbreeding.
  • the presently disclosed subject matter thus also provides methods for selecting a plant of the genus Glycine exhibiting resistance towards SBR comprising detecting in the plant the presence of one or more resistance alleles as defined herein.
  • the method comprises providing a sample of genomic DNA from a soybean plant; and (b) detecting in the sample of genomic DNA at least one molecular marker associated with resistance to SBR.
  • the detecting can comprise detecting one or more SNPs that are associated with resistance to SBR.
  • the providing of a sample of genomic DNA from a soybean plant can be performed by standard DNA isolation methods well known in the art.
  • the detecting of a molecular marker can in some embodiments comprise the use of one or more sets of primer pairs that can be used to produce one or more amplification products that are suitable markers for one of the SNPs.
  • a set of primers can comprise, in some embodiments, nucleotide sequences as set forth in SEQ ID NOs: 14-81.
  • the detecting of a molecular marker can comprise the use of a nucleic acid probe having a base sequence that is substantially complementary to the nucleic acid sequence defining the molecular marker and which nucleic acid probe specifically hybridizes under stringent conditions with a nucleic acid sequence defining the molecular marker.
  • a suitable nucleic acid probe can for instance be a single strand of the amplification product corresponding to the marker.
  • the detecting of a molecular marker is designed to discriminate whether a particular allele of an SNP is present or absent in a particular plant.
  • the presently disclosed methods can also include detecting an amplified DNA fragment associated with the presence of a particular allele of an SNP.
  • the amplified fragment associated with a particular allele of an SNP has a predicted length or nucleic acid sequence
  • detecting an amplified DNA fragment having the predicted length or the predicted nucleic acid sequence is performed such that the amplified DNA fragment has a length that corresponds (plus or minus a few bases; e.g., a length of one, two or three bases more or less) to the expected length as based on a similar reaction with the same primers with the DNA from the plant in which the marker was first detected or the nucleic acid sequence that corresponds (i.e., has a homology of in some embodiments more than 80%, in some embodiments more than 90%, in some embodiments more than 95%, in some embodiments more than 97%, and in some embodiments more than 99%) to the expected sequence as based on the sequence of the marker associated with that SNP in the plant in which the marker was
  • markers e.g., SNP alleles
  • trans-markers markers that are absent in resistant plants, while they were present in the susceptible parent(s)
  • the detecting of an amplified DNA fragment having the predicted length or the predicted nucleic acid sequence can be performed by any of a number or techniques, including but not limited to standard gel-electrophoresis techniques or by using automated DNA sequencers. The methods are not described here in detail as they are well known to the skilled person, although exemplary approaches are set forth in the EXAMPLES.
  • Molecular markers are used for the visualization of differences in nucleic acid sequences. This visualization can be due to DNA-DNA hybridization techniques after digestion with a restriction enzyme (e.g., an RFLP) and/or due to techniques using the polymerase chain reaction (e.g., STS, SSR/microsatellites, AFLP, and the like).
  • a restriction enzyme e.g., an RFLP
  • STS polymerase chain reaction
  • SSR/microsatellites e.g., SSR/microsatellites, AFLP, and the like.
  • all differences between two parental genotypes segregate in a mapping population based on the cross of these parental genotypes. The segregation of the different markers can be compared and recombination frequencies can be calculated.
  • mapping markers in plants are disclosed in, for example, Glick & Thompson (1993) Methods in Plant Molecular Biology and Biotechnology , CRC Press, Boca Raton, Fla., United States of America; Zietkiewicz et al. (1994) Genomics 20:176-183.
  • the recombination frequencies of molecular markers on different chromosomes and/or in different linkage groups are generally 50%. Between molecular markers located on the same chromosome or in the same linkage group, the recombination frequency generally depends on the distance between the markers. A low recombination frequency typically corresponds to a low genetic distance between markers on a chromosome. Comparing all recombination frequencies results in the most logical order of the molecular markers on the chromosomes or in the linkage groups. This most logical order can be depicted in a linkage map.
  • a group of adjacent or contiguous markers on the linkage map that is associated with an increased level of resistance to a disease; e.g., to a reduced incidence of acquiring the disease upon infectious contact with the disease agent and/or a reduced lesion growth rate upon establishment of infection, can provide the position of a locus associated with resistance to that disease.
  • the markers disclosed herein can be used in various aspects of the presently disclosed subject matter as set forth hereinbelow. Aspects of the presently disclosed subject matter are not to be limited to the use of the markers identified herein, however. It is stressed that the aspects can also make use of markers not explicitly disclosed herein or even yet to be identified.
  • the genetic unit “QTL” denotes a region of the genome that is directly related to a phenotypic quantifiable trait.
  • the markers provided by the presently disclosed subject matter can be used for detecting the presence of one or more SBR resistance alleles of the presently disclosed subject matter in a suspected SBR-resistant soybean plant, and can therefore be used in methods involving marker-assisted breeding and selection of SBR resistant soybean plants.
  • detecting the presence of a particular allele of an SNP of the presently disclosed subject matter is performed with at least one of the markers for the resistance loci defined herein.
  • the presently disclosed subject matter therefore relates in another aspect to a method for detecting the presence of a particular allele associated with SBR resistance, comprising detecting the presence of a nucleic acid sequence of the SNP in a suspected SBR-resistant soybean plant, which presence can be detected by the use of the disclosed markers and oligonucleotides.
  • the nucleotide sequence of an SNP of the presently disclosed subject matter can for instance be resolved by determining the nucleotide sequence of one or more markers associated with the SNP and designing internal primers for the marker sequences that can be used to determine which allele of the SNP is present in the plant.
  • the method can also comprise providing a oligonucleotide or polynucleotide capable of hybridizing under stringent hybridization conditions to a particular nucleic acid sequence of an SNP, in some embodiments selected from the SNPs disclosed herein, contacting the oligonucleotide or polynucleotide with genomic nucleic acid (or a fragment thereof, including, but not limited to a restriction fragment thereof) of a suspected SBR-resistant soybean plant, and determining the presence of specific hybridization of the oligonucleotide or polynucleotide to the genomic nucleic acid (or the fragment thereof).
  • the method is performed on a nucleic acid sample obtained from the suspected SBR-resistant soybean plant, although in situ hybridization methods can also be employed.
  • in situ hybridization methods can also be employed.
  • one of ordinary skill in the art can design specific hybridization probes or oligonucleotides capable of hybridizing under stringent hybridization conditions to the nucleic acid sequence of the allele associated with SBR resistance and can use such hybridization probes in methods for detecting the presence of an SNP allele disclosed herein in a suspected SBR-resistant soybean plant.
  • the presently disclosed subject matter also relates to methods for producing an SBR-resistant soybean plant comprising performing a method for detecting the presence of an allele associated with resistance to SBR in a donor soybean plant according to the presently disclosed subject matter as described above, and transferring a nucleic acid sequence comprising at least one allele thus detected, or an SBR resistance-conferring part thereof, from the donor plant to an SBR-susceptible recipient soybean plant.
  • the transfer of the nucleic acid sequence can be performed by any of the methods described herein.
  • An exemplary embodiment of such a method comprises the transfer by introgression of the nucleic acid sequence from an SBR-resistant donor soybean plant into an SBR-susceptible recipient soybean plant by crossing the plants. This transfer can thus suitably be accomplished by using traditional breeding techniques.
  • SBR-resistance loci are introgressed in some embodiments into commercial soybean varieties using marker-assisted selection (MAS) or marker-assisted breeding (MAB).
  • MAS and MAB involves the use of one or more of the molecular markers for the identification and selection of those offspring plants that contain one or more of the genes that encode for the desired trait.
  • identification and selection is based on selection of SNP alleles of the presently disclosed subject matter or markers associated therewith.
  • MAB can also be used to develop near-isogenic lines (NIL) harboring the QTL of interest, allowing a more detailed study of each QTL effect and is also an effective method for development of backcross inbred line (BIL) populations.
  • NIL near-isogenic lines
  • BIL backcross inbred line
  • a donor soybean plant that exhibits resistance to SBR and comprising a nucleic acid sequence encoding SBR resistance is crossed with an SBR-susceptible recipient soybean plant that in some embodiments exhibits commercially desirable characteristics, such as, but not limited to, disease resistance, insect resistance, valuable nutritional characteristics, and the like.
  • the resulting plant population (representing the F1 hybrids) is then self-pollinated and set seeds (F2 seeds).
  • the F2 plants grown from the F2 seeds are then screened for resistance to SBR.
  • the population can be screened in a number of different ways.
  • the population can be screened using a traditional disease screen. Such disease screens are known in the art.
  • a quantitative bioassay is used.
  • marker-assisted breeding can be performed using one or more of the herein-described molecular markers to identify those progeny that comprise a nucleic acid sequence encoding for SBR resistance. Other methods, referred to hereinabove by methods for detecting the presence of an allele associated with SBR resistance, can be used. Also, marker-assisted breeding can be used to confirm the results obtained from the quantitative bioassays, and therefore, several methods can also be used in combination.
  • Inbred SBR-resistant soybean plant lines can be developed using the techniques of recurrent selection and backcrossing, selfing, and/or dihaploids, or any other technique used to make parental lines.
  • SBR resistance can be introgressed into a target recipient plant (the recurrent parent) by crossing the recurrent parent with a first donor plant, which differs from the recurrent parent and is referred to herein as the “non-recurrent parent”.
  • the recurrent parent is a plant that is non-resistant or has a low level of resistance to SBR and possesses commercially desirable characteristics, such as, but not limited to (additional) disease resistance, insect resistance, valuable nutritional characteristics, and the like.
  • the non-recurrent parent exhibits SBR resistance and comprises a nucleic acid sequence that encodes SBR resistance.
  • the non-recurrent parent can be any plant variety or inbred line that is cross-fertile with the recurrent parent.
  • the progeny resulting from a cross between the recurrent parent and non-recurrent parent are backcrossed to the recurrent parent.
  • the resulting plant population is then screened for the desired characteristics, which screening can occur in a number of different ways. For instance, the population can be screened using phenotypic pathology screens or quantitative bioassays as known in the art.
  • the population can be screened using phenotypic pathology screens or quantitative bioassays as known in the art.
  • MAB can be performed using one or more of the hereinbefore described molecular markers, hybridization probes, or polynucleotides to identify those progeny that comprise a nucleic acid sequence encoding SBR resistance.
  • MAB can be used to confirm the results obtained from the quantitative bioassays.
  • the markers defined herein are suitable to select proper offspring plants by genotypic screening.
  • the F1 hybrid plants that exhibit an SBR-resistant phenotype or, in some embodiments, genotype and thus comprise the requisite nucleic acid sequence encoding SBR resistance are then selected and backcrossed to the recurrent parent for a number of generations in order to allow for the soybean plant to become increasingly inbred. This process can be performed for two, three, four, five, six, seven, eight, or more generations.
  • the progeny resulting from the process of crossing the recurrent parent with the SBR-resistant non-recurrent parent are heterozygous for one or more genes that encode SBR resistance.
  • a method of introducing a desired trait into a hybrid soybean variety can comprise:
  • the last backcross generation can be selfed in order to provide for homozygous pure breeding (inbred) progeny for SBR resistance.
  • inbred pure breeding
  • the result of recurrent selection, backcrossing, and selfing is the production of lines that are genetically homogenous for the genes associated with SBR resistance, and in some embodiments as well as for other genes associated with traits of commercial interest.
  • the development of a hybrid soybean variety in a soybean plant breeding program can, in some embodiments, involve three steps: (1) the selection of plants from various germplasm pools for initial breeding crosses; (2) the selfing of the selected plants from the breeding crosses for several generations to produce a series of inbred lines, which, individually breed true and are highly uniform; and (3) crossing a selected variety with an different variety to produce the hybrid progeny (F1). After a sufficient amount of inbreeding successive filial generations will merely serve to increase seed of the developed inbred.
  • an inbred line comprises homozygous alleles at about 95% or more of its loci.
  • a SBR-resistant soybean plant, or a part thereof, obtainable by a method of the presently disclosed subject atter is an aspect of the presently disclosed subject matter.
  • the SBR-resistant soybean plants of the presently disclosed subject matter, or part thereof, can be heterozygous or homozygous for the resistance traits (in some embodiments, homozygous).
  • the SBR resistance loci of the presently disclosed subject matter, as well as resistance-conferring parts thereof, can be transferred to any plant in order to provide for an SBR-resistant plant, the methods and plants of the presently disclosed subject matter are in some embodiments related to plants of the genus Glycine.
  • SBR-resistant soybean lines described herein can be used in additional crossings to create SBR-resistant plants.
  • a first SBR-resistant soybean plant of the presently disclosed subject matter can be crossed with a second soybean plant possessing commercially desirable traits such as, but not limited to, disease resistance, insect resistance, desirable nutritional characteristics, and the like.
  • this second soybean line is itself SBR-resistant.
  • this line is heterozygous or homozygous for one or more of the disclosed SBR resistance loci, in order for one or more of these traits to be expressed in the hybrid offspring plants.
  • Another aspect of the presently disclosed subject matter relates to a method of producing seeds that can be grown into SBR-resistant soybean plants.
  • the method comprises providing a SBR-resistant soybean plant of the presently disclosed subject matter, crossing the SBR-resistant plant with another soybean plant, and collecting seeds resulting from the cross, which when planted, produce SBR-resistant soybean plants.
  • the method comprises providing a SBR-resistant soybean plant of the presently disclosed subject matter, crossing the SBR-resistant plant with a soybean plant, collecting seeds resulting from the cross, regenerating the seeds into plants, selecting SBR-resistant plants by any of the methods described herein, self-pollinating the selected plants for a sufficient number of generations to obtain plants that are fixed for an allele associated with SBR-resistance in the plants, backcrossing the plants thus produced with soybean plants having desirable phenotypic traits for a sufficient number of generations to obtain soybean plants that are SBR-resistant and have desirable phenotypic traits, and collecting the seeds produced from the plants resulting from the last backcross, which when planted, produce soybean plants which are SBR-resistant.
  • a system for developing germplasm that has more than one mode of action against the fungus is made possible.
  • the use of this dual mode of action will assist in inhibiting the fungus from developing resistance.
  • a regime for inhibiting fungal resistance can include a number of different modes of action.
  • the different modes of action in an inhibiting fungus method could be through germplasm or seed treatments or chemical applications.
  • a system for inhibiting fungal resistance includes germplasm with one or more vertical resistant traits, and/or germplasm with one or more horizontal tolerance traits with the possible options of including a seed treatment that is active against the fungus, and/or an antifungal spray.
  • Antifungal sprays or treatments can include known antifungal compounds for this fungus but can also include glufosinate or glyphosate sprays, separate or in combination which also have an antifungal affect.
  • the regime for inhibiting antifungal resistance is very important for the continued effectiveness of germplasm resistance and chemical activity.
  • soybeans are presently produced which have fungal isolates that are no longer negatively affected by at least two of the known Rpp resistance alleles.
  • the ability of these fungal isolates to evolve so quickly could render entire soybean growing areas unprofitable unless the embodiments of the presently disclosed subject matter that provide soybean seeds which are protected by the SNP selected traits for a dual mode of action plus the optional seed treatment, or chemical applications is implemented.
  • SSR markers which are associated with soybean rust resistant qualitative and quantitative genes Rpp1, Rpp2, Rpp3, Rpp4, and Rpp5. This SSR information was employed to identify SNP markers that map to the regions of qualitative genes and quantitative trait loci (QTL) regions defined for each of the Rpp1, Rpp2, Rpp3, Rpp4, and Rpp5 genes.
  • QTL quantitative trait loci
  • the identified SNPs were validated on soybean rust resistant and susceptible lines. Analyses indicated that these SNPs mapped more closely and showed better associations to the Rpp gene than did the SSRs.
  • the information of validated SNPs for soybean rust is new and is used for appropriate breeding programs.
  • Rpp1, Rpp2, Rpp3, Rpp4, and Rpp5 genes were identified for the Rpp1, Rpp2, Rpp3, Rpp4, and Rpp5 genes, and the resistance gene first identified in the Japanese cultivar, Hyuuga. Hyten et al., 2007 used PI200492 to map Rpp1 to linkage group (LG) G between the SSR markers Sct — 187 and Sat — 064.
  • Rpp2 was mapped in PI230970 to the region on LG J between Sat — 255 and Satt620.
  • Rpp4 was mapped in PI459025 on LG G between SSR Satt288 and RFLP marker A885 — 1 (Silva et al. 2008, supra).
  • the cultivar Hyuuga (PI506764) was once thought to contain Rpp3 (the resistance gene contained in PIs 462312 and 459025B), but it is now believed that there are 2 separate rust resistance genes located near each other on LG C2, flanked by SSRs Satt460 and Sat — 263.
  • Rpp5 was recently identified in several lines (PI200487, PI200526, PI471904, PI200456) by Garcia et al., 2008, and mapped to LG N in a region flanked by the SSRs Sat — 275 and Sat — 280. The approximate positions of these genes and various markers are depicted in FIGS. 1A-1E .
  • the soybean linkage map developed by scientists at the USDA and made publicly available in 2006 can be found through the website of the United States Department of Agriculture (USDA) and is discussed in Choi et al. (2007) Genetics 176:685-696. This map was used to locate the flanking SSRs associated with each rust resistance gene and to select SNPs that were mapped within and close to each region. The polymorphism information and genomic sequence on either side of the SNP was used to design PCR-based assays to detect each allele. The sequences with the SNP indicated were either submitted to the Applied Biosystems Inc.
  • a total of 18 screening panel DNAs isolated from resistant lines (PI547875; PI200492; PI594538A; PI368039; PI547878; PI230970; PI224270; PI462312; PI578457A; PI518772; PI628932; PI506764; PI547879; PI459025B; PI200456; PI200526; PI200487; and PI471904) were used for the assays.
  • a PCR assay included a forward and reverse primer and a specific, fluorescent, dye-labeled probe for each of two alleles for a given SNP.
  • the probes contained different fluorescent reporter dyes (VIC®; Applied Biosystems, Inc., Foster City, Calif., United States of America; and 6-carboxyfluorescein-aminohexyl amidite (FAM), or N-TET-6-Aminohexanol (TET) and FAM) to differentiate the amplification of each allele.
  • a non-fluorescent quencher on each probe suppressed the fluorescence until amplification by PCR.
  • each probe annealed specifically to complementary sequences between the forward and reverse primer sites. Taq polymerase then cleaved the probes that were hybridized to each allele. Cleavage separated the reporter dye from the quencher, which resulted in increased fluorescence by the reporter dye.
  • the fluorescent signals generated by PCR amplification indicated that one or both alleles were present in the sample.
  • the probes also contained a minor groove binder at the 3′ end which resulted in increased melting temperatures (Tm), thereby allowing high specificity with the use of shorter oligos. These probes therefore exhibited greater Tm differences when hybridized to matched and mismatched templates, which provided more accurate allelic discriminations.
  • Probes of this type were manufactured at either ABI (MGBTM quencher) or Biosearch Technologies (BHQPLUSTM quencher). At the end of PCR thermal cycling, fluorescence of the two reporter dyes was measured on an ABI 7900. An increase in fluorescence for one dye only indicated homozygosity for the corresponding allele. An increase in both fluorescent signals indicated heterozygosity.
  • Tables 3-8 Exemplary starting lines and haplotypes determined using this method are presented in Tables 3-8.
  • “H” indicates that the line was heterozygous at that position
  • “ ⁇ ” indicates that the nucleotide at that position was not determined.
  • plants were selected based on their known phenotypic status and compared to the genotype at the specific SNP location.
  • DNA isolated from leaf tissue of seedlings 7-10 days after planting was diluted in TE buffer and stored at 4° C. until used in PCR reactions as described below.
  • PCR plates were placed in an ABI 9700 thermal cycler and the following program was run: an initial denaturation of 50° C. for 2 minutes followed by 95° C. for 10 minutes; 40 cycles of 95° C. for 15 seconds/60° C. for 1 minute; and a final elongation of 72° C. for 5 minutes. After the cycling, the samples were incubated at 4° C. until needed.
  • the ABI 7900 Sequence Detection System (or TAQMAN®) was used to visualize the results of an allelic discrimination (SNP) assay.
  • SNP allelic discrimination
  • SDS Sequence Detection System

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US20100192247A1 (en) 2010-07-29
US11206776B2 (en) 2021-12-28
WO2010009404A8 (fr) 2012-01-26
WO2010009404A2 (fr) 2010-01-21
US10070602B2 (en) 2018-09-11
US20160073598A1 (en) 2016-03-17
US20190166779A1 (en) 2019-06-06
US20200229366A1 (en) 2020-07-23
AR072521A1 (es) 2010-09-01

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